pharmaceutics
ArticleBiological Distribution after Oral Administration ofRadioiodine-Labeled Acetaminophen to Estimate
Gastrointestinal Absorption Function via OATPs,
OATs, and/or MRPs
Kakeru Sato 1,† , Asuka Mizutani 2,† , Yuka Muranaka 1 , Jianwei Yao 1 , Masato Kobayashi 2, * , Kana Yamazaki 3 ,Ryuichi Nishii 3 , Kodai Nishi 4 , Takeo Nakanishi 5 , Ikumi Tamai 6 and Keiichi Kawai 2,7
1

2

3

4

5
6

7

*
†

Citation: Sato, K.; Mizutani, A.;
Muranaka, Y.; Yao, J.; Kobayashi, M.;
Yamazaki, K.; Nishii, R.; Nishi, K.;
Nakanishi, T.; Tamai, I.; et al.
Biological Distribution after Oral
Administration ofRadioiodine-Labeled Acetaminophen
to Estimate Gastrointestinal
Absorption Function via OATPs,
OATs, and/or MRPs. Pharmaceutics
2023, 15, 497. https://doi.org/
10.3390/pharmaceutics15020497
Academic Editor: Leonard I. Wiebe
Received: 31 December 2022
Revised: 28 January 2023
Accepted: 30 January 2023

Division of Health Sciences, Graduate School of Medical Sciences, Kanazawa University, 5-11-80 Kodatsuno,
Kanazawa 920-0942, Japan
Faculty of Health Sciences, Institute of Medical, Pharmaceutical and Health Sciences, Kanazawa University,
5-11-80 Kodatsuno, Kanazawa 920-0942, Japan
Department of Molecular Imaging and Theranostics, Institute for Quantum Medical Science, Quantum Life
and Medical Science Directorate, National Institutes for Quantum Science and Technology, 4-9-1 Anagawa,Inage 263-8555, Japan
Department of Radioisotope Medicine, Atomic Bomb Disease Institute, Nagasaki University, 1-12-4 Sakamoto,Nagasaki 852-8523, Japan
Faculty of Pharmacy, Takasaki University of Health and Welfare, 60 Nakaorui-machi, Takasaki 370-0033, Japan
Faculty of Pharmaceutical Sciences, Institute of Medical, Pharmaceutical and Health Sciences,
Kanazawa University, Kakuma, Kanazawa 920-1192, Japan
Biomedical Imaging Research Center, University of Fukui, 23-3 Matsuokashimoaizuki, Eiheiji 910-1193, Japan
Correspondence: kobayasi@mhs.mp.kanazawa-u.ac.jp; Tel.: +81-76-265-2500
These authors contributed equally to this work.

Abstract: We evaluated the whole-body distribution of orally-administered radioiodine-125 labeledacetaminophen (125 I-AP) to estimate gastrointestinal absorption of anionic drugs. 125 I-AP wasadded to human embryonic kidney (HEK)293 and Flp293 cells expressing human organic aniontransporting polypeptide (OATP)1B1/3, OATP2B1, organic anion transporter (OAT)1/2/3, or carnitine/organic cation transporter (OCTN)2, with and without bromosulfalein (OATP and multidrug
resistance-associated protein (MRP) inhibitor) and probenecid (OAT and MRP inhibitor). The biological distribution in mice was determined by oral administration of 125 I-AP with and without
bromosulfalein and by intravenous administration of 125 I-AP. The uptake of 125 I-AP was significantly
higher in HEK293/OATP1B1, OATP1B3, OATP2B1, OAT1, and OAT2 cells than that in mock cells.
Bromosulfalein and probenecid inhibited OATP- and OAT-mediated uptake, respectively. Moreover,
125 I-AP was easily excreted in the urine when administered intravenously. The accumulation of
125 I-AP was significantly lower in the blood and urinary bladder of mice receiving oral administrationof both 125 I-AP and bromosulfalein than those receiving only 125 I-AP, but significantly higher inthe small intestine due to inhibition of OATPs and/or MRPs. This study indicates that whole-body
distribution after oral 125 I-AP administration can be used to estimate gastrointestinal absorption in
the small intestine via OATPs, OATs, and/or MRPs by measuring radioactivity in the urinary bladder.

Published: 2 February 2023

Keywords: gastrointestinal absorption; anion drugs and medicines; 125 I-acetaminophen; oral
administration; drug transporters
Copyright: © 2023 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms andconditions of the Creative Commons
Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

1. Introduction
Many oral drugs and medicines are absorbed into the blood via drug transporters
expressed on small intestinal epithelial cells and accumulate in the target tissues andorgans. Drug transporters are broadly divided into the solute carrier (SLC) and ATPbinding cassette (ABC) transporters, which contribute significantly to the influx and efflux

Pharmaceutics 2023, 15, 497. https://doi.org/10.3390/pharmaceutics15020497

https://www.mdpi.com/journal/pharmaceutics

Pharmaceutics 2023, 15, 497

Many oral drugs and medicines are absorbed into the blood via drug tran
expressed on small intestinal epithelial cells and accumulate in the target tissues
gans. Drug transporters are broadly divided into the solute carrier (SLC) and ATP
cassette (ABC) transporters, which contribute significantly to the influx anddrugs [1,2]. In the small intestine, these transporters are expressed on2 ofthe
9 villou
the brush border membrane and the vascular side of the basolateral membrane [3mainly involved in the absorption of oral drugs or medicines by the gastrointesti
ofThe
drugs
[1,2].
In the small intestine,these transporters
are expressedon thevillous1)side
SLC
transporters
on the brushborder membraneside(Figurecompriseof the brush border membrane and the vascular side of the basolateral membrane [3] andanion transporting polypeptide (OATP), mainly OATP2B1 (SLC21A9), and org
are mainly involved in the absorption of oral drugs or medicines by the gastrointestiion/carnitine(OCTN),
mainlyOCTN2 (SLC22A5)
peptide tranal
tract. The SLCtransportertransporters on
the brushborder membraneside (Figure 1)andcompriseorganic
anion
transporting
polypeptide(OATP),OATP2B1(SLC21A9),
and or(PEPT)1(SLC15A1)[4–7].
In addition,
onmainly
the brushbordermembraneside, AB
ganic
cation/carnitinetransporter(OCTN),
mainly
OCTN2(SLC22A5)
andpeptidetransporters comprise P-glycoprotein and multidrug resistance-associated protein
porter (PEPT)1 (SLC15A1) [4–7]. In addition, on the brush border membrane side, ABC
whereas, on the basolateral membrane side, these receptors express MRP3 [1,2,5transporters comprise P-glycoprotein and multidrug resistance-associated protein (MRP)2,
fore, thelevelsof thesea crucialrole inthe absorptio
whereas,onexpression
the basolateral
membraneside,transporters
these receptorsplay
express
MRP3 [1,2,5].
Therefore,
drugs
or medicines.
In transporters
addition, gastrointestinalthat alter
expressio
theexpression
levels of theseplay a crucial role indiseases
the absorptionof oralthe
drugsor
medicines.
In
addition,gastrointestinal
diseases
that
alter
the
expression
levelsoftheseof these transporters in small intestinal epithelial cells may reduce the gastrointes
transporters
smalldrugs
intestinal
epithelial cells may reduce the gastrointestinal absorption
sorption ofinoralor medicines.
of oral drugs or medicines.

Figure 1. Solute carrier and ATP-binding cassette transporters are mainly expressed on small inFigureepithelial
1. Soluteand ATP-binding
cassettetransporters
are mainly
on sm
testinal
cellcarrier
membranes.
OATP, organic aniontransporting
polypeptide;
OCTN,expressedorganic
testinal
epithelialcellmembranes.
OATP,
organic
anion
transporting
polypeptide;
OCTN
cation/carnitine transporter; PEPT, peptide transporter; MRP, multidrug resistance-associated protein.

cation/carnitine transporter; PEPT, peptide transporter; MRP, multidrug resistance-associ
The gastrointestinal absorption function is primarily measured using liquid chroprotein.

matography/mass spectrometry (LC/MS) [8] or LC/MS/MS. However, this technique
is limited because it requires liquid samples (e.g., blood) from the human body after the
The gastrointestinal
absorption
is primarily
using liquid
administrationof oral drugs or medicines
andfunction
cannot accuratelyreflect measured
the gastrointestinal
tography/mass
spectrometry(LC/MS)[8]or
LC/MS/MS.
However,
this
techniquabsorption function for specific SLC transporters on the brush border membrane in thesmallintestine. it
Therefore,
is asamples
need for an
imagingtechniquethathumancan accurately
ited because
requiresthere
liquid(e.g.,
blood)
from thebody after th
estimate
gastrointestinal
absorptionfunctiontopersonalize
drugadministration.
ouristration of oral drugs or medicines and cannot accurately reflect theIngastrointes
previous study, we used oral administration of iodine-123 labeled m-iodobenzylguanidine
sorption function for specific SLC transporters on the brush border membrane in t
(123 I-MIBG) to estimate gastrointestinal absorption of cationic anticancer drugs or medicines
intestine.
Therefore,
is aintestineneed forimagingtechniquecan liver,accurately
via
OCTN and/or
OCT inthere
the smallby anmeasuringradioactivity
in thatthe heart,andurinary bladderabsorption[9]. However,this imaging
method cannot
estimate
the absorptionIn our pgastrointestinal
function
to personalize
drugadministration.
functionoforganic
anion
drugs
and
medicines
inthegastrointestinal
tract.
study, we used oral administration of iodine-123 labeled m-iodobenzylguanid
Acetaminophen (AP, N-acetyl-p-aminophenol, and paracetamol) is an anionic drug
MIBG)
to estimate gastrointestinal absorption of cationic anticancer drugs or mthat is frequently used as an analog and antipyretic drug. AP is taken up by epithelial
via of
OCTN
and/or OCTtract
in theintestineby measuring
radioactivity
in the hea
cells
the gastrointestinal
via small
OATP andOAT [10].
We have previously
developed125
and urinary bladderimaging
methodestimate
radioiodine-125-labeledAP[9].
( However,I-AP) for the thisimagingof melanoma
andcannot
hypothesizedthat the ab
125 I-AP might also have affinities for OATP and/or OAT. Therefore, in the present study,function of organic anion drugs and medicines in the gastrointestinal tract.
125
we evaluatedthe whole-body
distributionof orally-administeredand
radioiodine-labeled
Acetaminophen(AP,
N-acetyl-p-aminophenol,
paracetamol) is I-an anio
AP to estimate gastrointestinal absorption of anionic drugs or medicines. This imaging

that is frequently used as an analog and antipyretic drug. AP is taken up by epitheof the gastrointestinal tract via OATP and OAT [10]. We have previously dePharmaceutics 2023, 15, 497

3 of 9technique may help estimate the gastrointestinal absorption of orally administered anionicdrugs or medicines, thereby personalizing drug administration.
2. Materials and Methods
2.1. 125 I Labeling of AP and Purification
125 I-AP was synthesized according to a previously reported protocol [11].

Briefly,

125 I-AP was synthesized using the chloramine T method by adding 4 mM chloramine T

and Na125 I to 10 mM AP. 125 I-AP was purified using high-performance liquid chromatography (HPLC, Hitachi, Ibaraki, Japan). Labeling rates were determined using thin-layer
chromatography (TLC, silica gel 60 F254; Millipore-Sigma, Burlington, MA, USA). Radiochemical purity was examined with HPLC.
2.2. HEK293 and Flp293 Cells Expressing High Levels of Various SLC Transporters
Human embryonic kidney (HEK)293 cells (American Type Culture Collection, Manassas, VA, USA) with high expression levels of OATP1B1 (SLC21A6, NM_006446), OATP1B3(SLC21A8, NM_019844), OATP2B1 (NM_007256), OAT2 (SLC22A7, NM_006672), andOCTN2 (NM_003060) were used. Flp293 cells derived from HEK293 cells stably express thehuman α1A-adrenoreceptor, with high expression of OAT1 (SLC22A6, NM_004790) andOAT3 (SLC22A8, NM_004254), were also used. HEK293/OATP1B1, HEK293/OATP1B3,
HEK293/OATP2B1, HEK293/OAT2, HEK293/OCTN2, Flp293/OAT1, Flp293/OAT3, andmock control cell lines were established by transfecting HEK293 and Flp293 cells withplasmids encoding each transporter. All cells were cultivated in Dulbecco’s Modified
Eagle’s medium (FUJIFILM Wako Chemicals, Osaka, Japan) mixed with 10% fetal bovine
serum (Life Technologies, Carlsbad, CA, USA), 1% sodium pyruvate, and 1% penicillin at
37 ◦ C and 5% CO2 .
2.3. Accumulation of 125 I-AP in HEK293 Cells
Before the experiments with 125 I-AP, OATP and OAT expressions were confirmed
using p-14 C-aminohippuric acid (PerkinElmer Inc., Waltham, MA, USA) in HEK293 andFlp293 cells, and OCTN2 expression was confirmed using methyl-3 H-4-phenylpyridinium(American Radiolabeled Chemicals Inc., St. Louis, MO, USA) in HEK293 cells [9]. HEK293
cells expressing the respective SLC transporter were seeded at 1.0 × 105 cells/well in
12-well plastic plates in DMEM and cultivated at 37 ◦ C and 5% CO2 for 24 h. On the day ofthe experiments, each cell type was pre-incubated in phosphate-buffered saline (PBS) for
5 min and then incubated with 125 I-AP (18.5 kBq/well) for 5 min (n = 4). After incubation,
the cells were washed twice with 600 µL of PBS and lysed with 500 µL of 0.1 M NaOH.
Radioactivity was measured using a gamma counter (AccuFLEX γ7000; Hitachi Aloka
Medical, Tokyo, Japan). Intracellular protein levels were measured using a bicinchoninicacid protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA), using bovine serum
albumin as the standard.
2.4. Accumulation of 125 I-AP in HEK293 Cells Treated with Inhibitors of Drug Transporters
HEK293 cells expressing the respective SLC transporter were seeded at 1.0 × 105 cells/wellin 12-well plastic plates in DMEM and cultivated at 37 ◦ C and 5% CO2 for 24 h. The cells
were pre-incubated in PBS for 5 min and then incubated with 125 I-AP (18.5 kBq/well) and
1.0 mM bromosulfalein (Sigma-Aldrich, St. Louis, MO, USA), an inhibitor of OATPs and
MRPs [12,13], or 1.0 mM probenecid (Sigma-Aldrich), an inhibitor of OATs and MRPs for
5 min (n = 4) [13,14]. After incubation, the cells were washed twice with 600 µL of PBS andlysed with 500 µL of 0.1 M NaOH. Radioactivity was measured using an auto well gamma
counter. Intracellular protein levels were measured using a bicinchoninic acid protein assay
kit (Thermo Fisher Scientific), using bovine serum albumin as the standard.

Pharmaceutics 2023, 15, 497

4 of 92.5. Biological Distribution of 125 I-AP in Normal Mice
All animal experiments performed in this study were conducted in compliance withthe ethical standards of Kanazawa University (Animal Care Committee of Kanazawa
University, AP-194046) and the international standards for animal welfare and institutional guidelines. 125 I-AP was orally and intragastrically administered to ddY mice (male,
8 weeks old; SLC Inc., Hamamatsu, Japan) that had fasted for 6 h, using Fuchigami animal
feeding needles (Natsume Seisakusho Co., Ltd., Tokyo, Japan) at a dose of approximately
37 kBq/200 µL per mouse. After 5, 10, 30, and 60 min of administration (n = 4), heart blood
samples were collected under isoflurane anesthesia (FUJIFILM Wako Pure Chemical Corporation), and mice were sacrificed by cervical dislocation. The organs (thyroid gland, heart,
stomach, liver, small intestine, kidney, and urinary bladder) were then quickly collected.
The weight of each organ was measured using an electronic balance (AUX220, Shimadzu
Corporation, Kyoto, Japan), and radioactivity was measured using an auto gamma counter(AccuFLEX gamma ARC-7010, Hitachi, Ltd., Tokyo, Japan). The results are shown as
the percent injected dose (%ID) for the thyroid gland, stomach, small intestine, and urinary bladder and %ID/g wet tissue (%ID/g tissue) for the other organs. As the thyroid
gland of mice is quite small and the stomach and small intestine can’t completely remove
the contents, it is difficult to measure the accurate weight of these organs. Thus, theseorgans are indicated by %ID. The urinary bladder results are expressed in %ID because
it is undesirable that the radioactivity of urine is corrected by the weight of the urinary
bladder. In addition, 125 I-AP was diluted with saline to approximately 20 kBq/200 µL
and administrated intravenously through the tail vein of the mice. After 5, 10, 30, and60 min of administration (n = 4), heart blood samples were collected, and their weights andradioactivity were measured.
2.6. Biological Distribution of 125 I-AP Administered with an OATP Inhibitor
Mice (ddY, male, 8 weeks old; SLC Inc., Hamamatsu, Japan) fasted for 6 h before
using Fuchigami feeding needles for oral and intragastric administration of the drugs.
125 I-AP was diluted with saline to approximately 20 kBq/100 µL, and 100 µL of 125 I-AP
was orally administered to mice using feeding needles after 5 min of oral administrationof bromosulfalein, an inhibitor of OATP. After 5, 10, 30, and 60 min of administration(n = 4), heart blood samples were collected under isoflurane anesthesia, and the mice weresacrificed by cervical dislocation. The organs (thyroid gland, heart, stomach, liver, small
intestine, kidney, and urinary bladder) were quickly collected. The weight of each organ
was measured using an electronic balance, and radioactivity was measured using a gamma
counter. The results are shown as %ID or %ID/g of tissue.
2.7. Statistical Analysis
P-values were analyzed using the two-tailed paired Student’s t-test for comparisons
between two groups and analysis of variance and the post hoc comparisons with Dunn’s
test for comparisons among three groups using GraphPad Prism 8 statistical software(GraphPad Software, Inc., La Jolla, CA, USA). Statistical significance was set as p < 0.01.
3. Results
3.1. 125 I-AP Accumulation in HEK293 and Flp293 Cells Expressing High Levels of Different
SLC Transporters
125 I-AP was labeled with an efficiency of more than 80% and was obtained with aradiochemical purity of more than 95% after HPLC analysis. Mass-spectrometry data of
127 I-AP have been reported in our previous study [11]. The date of 127 I-AP revealed an MH(+) of 278, which matched the calculated MH (+) of 278 C8 H8 INO2 .
The accumulation of 125 I-AP in HEK293 and Flp293 cells with high expression of
an SLC transporter is shown in Figure 2. Before performing the experiments with 125 IAP, the function of the SLC transporter in HEK293 and Flp293 cells was confirmed using 3 H- or 14 C-labeled substrates [9]. Accumulation of the p-14 C-aminohippuric acid

Pharmaceutics 2023, 15, 497

0.04%ID/mg protein for Flp293/OAT3.
Compared to HEK293 mock cells, the intracellular accumulation of 125I-AP was significantly increased in HEK293/OATP1B1 (p < 0.05), HEK293/OATP1B3 (p < 0.05),
HEK293/OATP2B1 (p < 0.01), and HEK293/OAT2 cells (p < 0.01). However, the accumulation of 125I-AP was not increased in HEK293/OCTN2 cells compared to that in HEK293
5 of 9mock cells. Compared to Flp-mock cells, the accumulation of 125I-AP was significantly increased in Flp293/OAT1 (p < 0.05) but not in Flp293/OAT3 cells. In the presence of bromosulfalein, an inhibitor of OATP and MRP, the accumulation of 125I-AP was significantly
into HEK293 mock cells is 0.19 ± 0.03%ID/mg protein for HEK mock for OATP and
decreased in HEK293/OATP1B1, HEK293/OATP1B3, and HEK293/OATP2B1 cells comOAT2 and into Flp293 mock cells is 0.25 ± 0.06%ID/mg protein for Flp-mock for OAT1
pared to that in HEK293 mock cells. In the presenceof probenecid, an inhibitor of OAT
and OAT3. Accumulation of the methyl-3 H-4-phenylpyridinium into HEK293 mock
125I-AP was significantly decreased in Flp293/OAT1,
and
MRP,
the
accumulationofcells is 0.09 ± 0.06%ID/mg protein for HEK mock for OCTN2. In addition, accumuFlp293/OAT3,
and3 H-4-phenylpyridinium
HEK293/OAT2 comparedthatin Flp293 or HEK293
mockcells; howlation of methylor to
p-14
C-aminohippuric
acid into
HEK293and125I-AP in HEK293/OAT2 cells did not decrease to the same level
ever,
the
accumulationofFlp293 cells expressing a SLC transporter is as follows: 0.28 ± 0.06%ID/mg protein for
125I-AP was signifias
that in HEK293 mockcells.
These results
suggestthat the uptake of 0.34
HEK293/OATP1B1,
0.27±
0.05%ID/mg
proteinfor HEK293/OATP1B3,± 0.09%ID/mg
cantlyin HEK293/OATP1B1,
HEK293/OATP1B3,
HEK293/OATP2B1,
protein higher
for HEK293/OATP2B1,
0.42 ± 0.11%ID/mg
protein for
HEK293/OAT2,
Flp293/OAT1,
and HEK293/OAT2
cells than that inmockcells, and thatbromosulfalein
0.39 ± 0.08%ID/mg
protein for HEK293/OCTN2,
0.46± 0.03%ID/mg
proteinfor Flp293/
andprobenecid
inhibited
OATPand
OAT-mediateduptake,respectively.
OAT1 and 0.45 ± 0.04%ID/mg protein for Flp293/OAT3.

Figure 2. Accumulation of 125 I-AP in HEK293 and Flp293 cells with high expression of a solute
FigureAccumulationof 125I-AP inand Flp293
cells
with
expressionof a solute
carrier 2.
transporter(n = 4).
BeforetheHEK293
experimentswith 125
I-AP,
thehighfunctionof the SLC
transporter
125I-AP, the function of the SLC transporter
carriertransporter(n=4).
Beforetheexperiments
within HEK293 and Flp293 cells was confirmed using 3 H- or 14 C-labeled substrates. Accumulation of
in HEK293
and Flp293 cells was confirmed using 3H- or 14C-labeled substrates. Accumulation of
the p-14 C-aminohippuric acid into HEK293 mock cells is 0.19 ± 0.03%ID/mg protein for HEK mockthe p-14C-aminohippuric acid into HEK293 mock cells is 0.19 ± 0.03%ID/mg protein for HEK mockfor OATP and OAT2 and into Flp293 mock cells is 0.25 ± 0.06%ID/mg protein for Flp-mock for
OAT1 and OAT3. Accumulation of the methyl-3 H-4-phenylpyridinium into HEK293 mock cells is
0.09 ± 0.06%ID/mg protein for HEK mock for OCTN2. In addition, accumulation of methyl-3 H-4phenylpyridinium or p-14 C-aminohippuric acid into HEK293 and Flp293 cells expressing a SLC transporter is as follows: 0.28 ± 0.06%ID/mg protein for HEK293/OATP1B1, 0.27± 0.05%ID/mg protein
for HEK293/OATP1B3, 0.34 ± 0.09%ID/mg protein for HEK293/OATP2B1, 0.42 ± 0.11%ID/mg protein for HEK293/OAT2, 0.39 ± 0.08%ID/mg protein for HEK293/OCTN2, 0.46 ± 0.03%ID/mg protein for Flp293/OAT1 and 0.45 ± 0.04%ID/mg protein for Flp293/OAT3. 125 I-AP accumulation is significantly increased in HEK293/OATP1B1, HEK293/OATP1B3, HEK293/OATP2B1, HEK293/OAT2,
and Flp293/OAT1 cells compared to that in mock cells. The addition of bromosulfalein, an inhibitor of OATP and MRP, inhibits 125 I-AP uptake into HEK293/OATP1B1, HEK293/OATP1B3,
HEK293/OATP2B1 cells. The addition of probenecid, an inhibitor of OAT and MRP, inhibits 125 I-AP
uptake into HEK293/OAT2 and Flp293/OAT1 cells. † p < 0.01 and * p < 0.05 vs. mock cells andcells loaded with both inhibitors. OATP, organic anion transporting polypeptide; OCTN, organic
cation/carnitine transporter; PEPT, peptide transporter; MRP, multidrug resistance-associated protein.

Compared to HEK293 mock cells, the intracellular accumulation of 125 I-AP was
significantly increased in HEK293/OATP1B1 (p < 0.05), HEK293/OATP1B3 (p < 0.05),
HEK293/OATP2B1 (p < 0.01), and HEK293/OAT2 cells (p < 0.01). However, the accumulation of 125 I-AP was not increased in HEK293/OCTN2 cells compared to that in HEK293
mock cells. Compared to Flp-mock cells, the accumulation of 125 I-AP was significantly
increased in Flp293/OAT1 (p < 0.05) but not in Flp293/OAT3 cells. In the presence of
bromosulfalein, an inhibitor of OATP and MRP, the accumulation of 125 I-AP was signifi-

Pharmaceutics 2023, 15, 497

6 of 9cantly decreased in HEK293/OATP1B1, HEK293/OATP1B3, and HEK293/OATP2B1 cells
compared to that in HEK293 mock cells. In the presence of probenecid, an inhibitor of
OAT and MRP, the accumulation of 125 I-AP was significantly decreased in Flp293/OAT1,
Flp293/OAT3, and HEK293/OAT2 compared to that in Flp293 or HEK293 mock cells;
however, the accumulation of 125 I-AP in HEK293/OAT2 cells did not decrease to thesame level as that in HEK293 mock cells. These results suggest that the uptake of 125 I-AP
was significantly higher in HEK293/OATP1B1, HEK293/OATP1B3, HEK293/OATP2B1,
Flp293/OAT1, and HEK293/OAT2 cells than that in mock cells, and that bromosulfalein
and probenecid inhibited OATP- and OAT-mediated uptake, respectively.
3.2. Biological Distribution of Orally-Administered 125 I-AP with or without Bromosulfalein and of
Intravenously-Administered 125 I-AP In Vivo
The in vivo stability of intravenously-administered 125 I-AP has been confirmed inblood and homogenized liver of mice using TLC analysis in our previous study [11].
Although 125 I-AP was slightly metabolized at 5 min after administration, it remained stable
60 min after administration. The biodistribution of orally-administered 125 I-AP with orwithout the oral administration of bromosulfalein and intravenously-administered 125 I-AP
is shown in Table 1. In all the mice, the accumulation of 125 I-AP in the thyroid gland was
low. In the mice group receiving an intravenous administration of 125 I-AP, 125 I-AP was
easily excreted in the urine. In contrast, the urinary excretion of 125 I-AP was slower inthe oral administration group than in the intravenous administration group. In addition,compared to mice receiving oral administration of 125 I-AP, those receiving intravenous
administration showed a higher accumulation of 125 I-AP in the blood, heart, small intestine,
kidney, and urinary bladder at 5 min after administration. In the group that received
oral administration of 125 I-AP and bromosulfalein, the accumulation of 125 I-AP in the
blood significantly decreased at 5 min after administration, and that in the urinary bladdersignificantly decreased at 5, 10, and 30 min after administration. However, in the small
intestine, the accumulation of 125 I-AP increased at 5 min after administration. Therefore,
biological distribution after oral 125 I-AP administration is used to estimate gastrointestinal
absorption in the small intestine by measuring radioactivity at the early time phase in theurinary bladder.
Table 1. Biological distribution of 125 I-AP in mice by oral administration, with or without bromosulfalein, and by intravenous administration.
Time after 125 I-AP Administration
Mice

Organ

5 min

10 min30 min60 minOraladministration

Blood
Thyroid glandHeart
StomachLiver
Small intestine
KidneyUrinary bladder

3.97 ± 0.58
0.22 ± 0.08
1.48 ± 0.46
62.7± 9.91
2.41 ± 0.82
4.39 ± 2.48
4.11 ± 0.25
0.23 ± 0.13

4.53 ± 0.71
0.04 ± 0.00
1.59 ± 0.16
38.5 ± 7.17
2.38 ± 0.75
15.6 ± 5.89
6.86 ± 2.98
2.22 ± 12.5

2.76 ± 0.68
0.09 ± 0.02
1.15 ± 0.33
22.9 ± 9.78
1.42 ± 0.27
16.6 ± 5.76
3.53 ± 0.39
17.8 ± 7.29

1.19 ± 0.30
0.13 ± 0.07
0.44 ± 0.15
15.9 ± 8.55
0.54 ± 0.13
19.6 ± 7.41
5.78 ± 4.38
16.6 ± 10.8

Oraladministration
withbromosulfalein

Blood
Thyroid glandHeart
StomachLiver
Small intestine
KidneyUrinary bladder

1.10 ± 0.33 *0.18 ± 0.09
1.53 ± 0.63
57.0 ± 35.1
1.81 ± 0.30
15.2 ± 6.85 *4.22 ± 1.44
0.06 ± 0.02 *3.38 ± 1.76
0.07 ± 0.03
1.25 ± 0.68
56.3 ± 23.2
1.75 ± 0.68
16.4 ± 2.79
5.50 ± 2.06
0.20 ± 0.23 *2.40 ± 0.78
0.08 ± 0.01
0.85 ± 0.28
24.8 ± 15.0
1.22 ± 0.31
19.0 ± 4.68
5.98 ± 2.42
7.20 ± 2.71 *1.11 ± 0.11
0.13 ± 0.02
0.43 ± 0.06
11.3 ± 1.79
0.64 ± 0.18
18.4 ± 5.79
2.01 ± 0.71
17.8 ± 0.59

Intravenous
administration

Blood
Thyroid gland7.95 ± 2.15 †0.05 ± 0.01 *3.51 ± 1.13
0.05 ± 0.04

1.55 ± 0.43
0.06 ± 0.02

0.79 ± 0.10 *0.10 ± 0.05

Pharmaceutics 2023, 15, 497

7 of 9

Table 1. Cont.
Time after 125 I-AP Administration
Mice

Organ

5 min

10 min30 min60 minHeart
StomachLiver
Small intestine
KidneyUrinary bladder2.99 ± 0.60 *1.04 ± 0.57 †2.81 ± 0.19
8.52 ± 0.99
19.4 ± 3.13 *10.60 ± 7.65 *1.14 ± 0.17
1.27 ± 0.30 †1.07 ± 0.08 †8.31 ± 0.88
12.1 ± 7.89
16.6 ± 19.0

0.49 ± 0.12 *1.01 ± 0.44 †0.56 ± 0.15 *8.56 ± 1.56 *6.85 ± 8.23
23.7 ± 22.0

0.26 ± 0.06 *1.05 ± 0.41 *0.32 ± 0.04 *9.21 ± 0.67 †4.30 ± 2.84
42.7 ± 16.7

%ID indicates the percent injected dose for the thyroid gland, stomach, small intestine, and urinary bladder.
%ID/g indicates the percent injected dose per gram of blood, heart, liver, and kidney. Values represent themean ± standard deviation of four mice. * p < 0.01 and † p < 0.05 compared to oral administration.

4. Discussion
The gastrointestinal absorption function varies among individuals and in the same
individual under disease states. Therefore, an imaging method that can estimate gastrointestinal absorption function would help personalize drug administration. In ourprevious study, we investigated the distribution of the oral administration of 123 I-MIBG
to estimate the gastrointestinal absorption of cationic anticancer drugs or medicines via
OCTN and OCT in the small intestine [9]. This imaging technique may help investigate
differences in gastrointestinal absorption, which can affect the activity of cationic anticancer
drugs. In the present study, we investigated the biodistribution of orally-administered
125 I-AP to estimate the absorption function of anionic anticancer drugs or medicines in thegastrointestinal tract.
AP is an anionic drug that is taken up by cells via OATP and OAT [10]. In the present
study, 125 I-AP had affinity for OATP1B1, OATP1B3, OATP2B1, OAT1, and OAT2. Moreover,in the presence of bromosulfalein, an OATP inhibitor, the accumulation of 125 I-AP in cellswith high expressions of OATPs and OAT1 decreased to the same levels as that in HEK293
or Flp293-mock cells (control). However, in the presence of probenecid, an inhibitor of OAT,
the accumulation of 125 I-AP in cells with high OAT2 expression levels did not decrease to thesame levels as those in the control cells. These results suggest that the effect of OATPs on theaccumulation of 125 I-AP was more significant than that of OAT2. However, bromosulfalein
inhibits not only OATPs but also OAT2 [15]. Therefore, the reduced accumulation of 125 IAP in HEK293/OATP1B1, HEK293/OATP1B3, and HEK293/OATP2B1 in the presence of
bromosulfalein may also partly involve OAT2.
Among SLC transporters present on the brush border membrane side of the small intestine, OATP2B1 is the most abundant, whereas OATs, including OAT2, are slightly expressedin the small intestine [4–7]. In the present study, after intravenous and oral administrationof 125 I-AP in mice, the accumulation of 125 I-AP in the thyroid gland was low, indicatingthat 125 I-AP was little de-iodine in vivo. In addition, intravenously-administrated 125 I-AP
showed high in vivo stability of 125 I-AP in the blood (94.1 ± 5.1% at 60 min of administration) and liver (88.2 ± 5.5% at 60 min of administration) of mice in our previous
study [11]. Therefore, we consider that 125 I-AP with oral administration also shows highin vivo stability.
Furthermore, compared to control mice, mice receiving oral administration of 125 I-AP
with bromosulfalein showed significantly decreased radioactivity in the blood 5 min afteradministration but significantly increased radioactivity in the small intestine. These results
suggest that OATP2B1 is the key transporter involved in the gastrointestinal absorption oforally-administered 125 I-AP because the uptake of 125 I-AP from the brush border membrane
to blood was inhibited by the administration of bromosulfalein.
Bromosulfalein also inhibits MRPs that are expressed on both sides of the brush border
membrane and basolateral membrane in the small intestine [13]. In addition, because thechemical structure of AP contains a peptide bond in its chemical structure, we evaluatedthe biological distribution of the oral administration of 125 I-AP with a peptide substrate,

Pharmaceutics 2023, 15, 497

8 of 9cephradine. The administration of cephradine increased the gastrointestinal absorption of
125 I-AP (average 9.61, 9.86, 8.96, 2.66 %ID/g in accumulation of blood at 5, 10, 30, 60 min

after oral administration with cephradine). Collectively, these results suggest that 125 I-AP
would be absorbed by OATPs and/or OATs on the brush border membrane and by MRP
on the basolateral membrane of the small intestine.
Anionic anticancer drugs or medicines are generally excreted via MRPs of the ABCtransporters. Therefore, 125 I-AP also may have an affinity for MRPs. In the present study,
the accumulation of 125 I-AP in the urinary bladder of mice receiving oral administrationof 125 I-AP and bromosulfalein was significantly decreased within 30 min compared tothat in mice orally administered only 125 I-AP. This effect is due to the inhibition of gastrointestinal absorption by bromosulfalein. Therefore, when gastrointestinal diseases thatalter the expression levels of these transporters in small intestinal epithelial cells reduce
the gastrointestinal absorption of oral drugs or medicines, it is possible to estimate thegastrointestinal absorption function of the small intestine via OATPs and/or OATs usingtime–radioactivity curves of the urinary bladder in the early time phase of 123 I-AP imaging,which was changed from 125 I to 123 I.
Currently, the gastrointestinal absorption function of orally administrated drugs
or medicines is primarily measured using LC/MS or LC/MS/MS [8]. We can confirm
pharmacokinetics with these data using the samples (blood etc.) after oral administrationof a drug or medicine [16]. However, the LC/MS or LC/MS/MS data are difficult to
use to evaluate the effect of the individual drug transporter in the small intestine on
gastrointestinal absorption. In addition, the absorption function becomes apparent after oral
drug or medicine administration. Whole-body imaging after oral 123 I-AP administration
cloud reflects the function of drug transporters prior to oral administration of the drugor medicine. Therefore, it may be possible to control the appropriate dose of the drug or
medicine for an individual patient.
We speculate that the effect of bromosulfalein is limited because we did not observea reduction in the accumulation of 125 I-AP in the liver that has high expression levels
of OATPs. When 123 I-MIBG whole-body imaging could estimate the gastrointestinal
absorption function via cationic transporters in our previous study, we used DSS-induced
experimental colitis mouse as well as the effect of inhibitors to drug transporters in thesmall intestine because DSS-induced experimental colitis mouse decreased expression
levels of OCTN1 [17]. However, there are no disease mouse models which decreased theexpression of OATP and/or OAT. If more effective mouse models, such as OATP- or OATdeficient mouse models, were used, the biological distribution after the oral administrationof 125 I-AP would be slightly different from that reported in the present study. In the future,
whole-body imaging after oral 123 I-AP administration should be performed to estimate
gastrointestinal absorption function via OATPs, OATs, and/or MRPs in the small intestine.
5. Conclusions
The present study demonstrates that whole-body distribution after oral 125 I-AP administration can be used to estimate gastrointestinal absorption of anionic drugs or medicines
in the small intestine via OATPs, OATs, and/or MRPs by measuring radioactivity in theurinary bladder. Before oral administration with a drug or medicine, whole-body imaging
after oral 123 I-AP administration could estimate the function of OATPs, OATs, and/or MRPs
in the small intestine and lead to control of the appropriate dose of orally administrated
drugs or medicines for an individual patient as personalized medicine.
Author Contributions: Conceptualization, M.K.; methodology, K.S., A.M., Y.M. and K.N.; investigation, K.S., A.M., Y.M., J.Y. and M.K.; resources, K.Y. and R.N.; writing—original draft preparation,
K.S.; writing—review and editing, M.K.; supervision, T.N., I.T., K.K. and M.K. All authors have readand agreed to the published version of the manuscript.

Pharmaceutics 2023, 15, 497

9 of 9Funding: This study was funded in part by Grants-in-Aid for Scientific Research from the Japan
Society for the Promotion of Science (No. 21H02865, 22K19504, 22H03016) and the Network-type
Joint Usage/Research Center for Radiation Disaster Medical Science.
Institutional Review Board Statement: Not applicable.
Informed Consent Statement: Not applicable.
Data Availability Statement: The authors confirm that the data supporting the findings of this study
are available within the article.
Acknowledgments: The authors would like to thank Mikie Ohtaki and other staff members of theFaculty of Health Sciences, Kanazawa University.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the designof the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; orin the decision to publish the results.

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